Natural killer cell function is regulated by activating and inhibitory receptors. The inhibitory receptors belong to the Ly49 family and the CD94/NKG2 families. Their ligands are classical and non-classical class I MHC molecules respectively. Each NK cell expresses at least one receptor that is inhibited by self MHC class I molecules. Thus, in adult mice, NK cells express a receptor repertoire that ensures self tolerance. The major goal of this project is to understand the regulation of NK cell receptor repertoire in developing NK cells. Most previous studies have focused on LY49 molecules, but studies conducted during the past funding period have revealed that CD94/NKG2 receptors are expressed earlier in ontogeny than the LY49 family, hence we will investigate the independent and co-regulation of these two families on developing NK cells. These studies are made possible because we have developed two culture systems-one derived from fetal liver and the other from marrow progenitors, in which receptor negative NK precursors can be induced to express class I receptors. Ly49 molecules, recognize classical class I molecules whereas CD94/NKG2 interact with non-classical Qa-1b molecules. By an analysis of class I knock out mice and Qa-1b knock out mice we will determine the role of these two types of MHC receptors on the development of NK receptors. By co-culture of receptor negative precursors and stromal cells from the knock out mice, we will determine how the presence or absence of classical and non-classical MHC molecules influence the acquisition and function of various receptor family members. The ability to generate Ly49+ and Ly49- in NK cells under defined conditions will allow us to determine the molecular signals that induce Ly49 gene expression. For this we will use differential m- RNA display and modified subtractive hybridization to define genes that influence Ly49 expression. Finally, we will generate antibodies against novel inhibitory receptors that are expressed on immature, developing NK cells, and also attempt to identify the ligands of such receptors. The experiments outlined here will allow a better understanding of how NK cell receptor development is regulated.